Vaccination is one of the most effective methods to prevent morbidity and mortality related to infectious diseases, yet there are many viral infections, such as Human Immunodeficiency Virus (HIV) and cytomegalovirus (CMV), for which durable, broadly cross-protective vaccines remain desperately needed. Although most of the focus in vaccine development has been on adaptive immune responses, natural killer (NK) cells are innate lymphocyte responders that rapidly and robustly kill virus-infected cells. Recent data has demonstrated that NK cells share features of adaptive immune responses-including the ability to respond more quickly and robustly upon a second challenge with the same antigen. Thus, they show great promise in establishing early control of infection in a vaccine or therapeutic setting where adaptive immune responses have proven to be too little and too late. Before this response can be elicited successfully, NK cell recognition of virus-infected cells must be understood at a detailed level. However, NK cells are a highly heterogeneous cell population, capable of a vast variety of functions and possessing a complex mosaic of surface receptors. The limits of fluorescence cytometry have prevented the simultaneous examination of all of these traits. To address this question, we propose the use of a novel technology, Cytometry by Time-Of-Flight (CyTOF). CyTOF can detect up to 40 cellular markers simultaneously, a considerable advance from the traditional fluorescence cytometry limit of 12-18. Thus, we will use CyTOF to comprehensively measure NK cell phenotype and functions during killing of virus-infected cells. We will define the specific populations of effector NK cells critical for responsesto HIV, influenza, and CMV by developing in vitro viral suppression assays in which NK cells from healthy donors recognize and kill autologous cells infected with these viruses. The highly parametric, deep profiling capabilities of CyTOF, applied to this specific, controlled interrogatio of antiviral NK cell function, will allow us to gain profound insight into which NK cells are activly recognizing and killing virus- infected cells. Results will be analyzed using a variety of complementary statistical techniques and dimensionality reduction algorithms. Once this analysis has defined specific subsets of NK cells involved in killing virus-infected cells, we will complement these findings using in vitro functional assays to define precisely how the NK cells are recognizing and killing the infected cells. These assays classically examine single receptors or functions, but will be adapted to simultaneously interrogate multiple receptors or functions in a combinatorial manner. Our innovative approach aims to understand which NK cell subsets to prime and which functions to stimulate in order to purposefully elicit an antiviral response in a vaccine or therapeutic setting. Our findings will therefore drive the development of innovative new approaches to vaccination.